Abstract
ABSTRACTEndocytosis of caveolae has previously been implicated in the repair of plasma membrane wounds. Here, we show that caveolin-1-deficient fibroblasts lacking caveolae upregulate a tubular endocytic pathway and have a reduced capacity to reseal after permeabilization with pore-forming toxins compared with wild-type cells. Silencing endophilin-A2 expression inhibited fission of endocytic tubules and further reduced plasma membrane repair in cells lacking caveolin-1, supporting a role for tubular endocytosis as an alternative pathway for the removal of membrane lesions. Endophilin-A2 was visualized in association with cholera toxin B-containing endosomes and was recruited to recently formed intracellular vacuoles containing Trypanosoma cruzi, a parasite that utilizes the plasma membrane wounding repair pathway to invade host cells. Endophilin-A2 deficiency inhibited T. cruzi invasion, and fibroblasts deficient in both caveolin-1 and endophilin-A2 did not survive prolonged exposure to the parasites. These findings reveal a novel crosstalk between caveolin-1 and endophilin-A2 in the regulation of clathrin-independent endocytosis and plasma membrane repair, a process that is subverted by T. cruzi parasites for cell invasion.
Highlights
For injured cells to survive, plasma membrane (PM) wounds must be rapidly repaired
Consistent with the extensive functional similarities previously identified between PM repair and T. cruzi invasion, we show that recruitment of EndoA2 to tubular PM invaginations plays a critical role in the mechanism by which the intracellular protozoan parasite T. cruzi invades host cells
Cav1 knockout mouse embryonic fibroblasts (MEFs) have reduced PM repair capacity To further investigate the mechanism of caveolae-independent PM repair detected in B cells (Miller et al, 2015), we used MEFs derived from WT and Cav1 KO littermate mice (Razani et al, 2001) and performed 5 min SLO wounding assays, followed by staining with the membrane-impermeable dye propidium iodide (PI) and flow cytometry analysis to assess the extent of PM repair (Idone et al, 2008b)
Summary
For injured cells to survive, plasma membrane (PM) wounds must be rapidly repaired. Wound repair is fast and critically dependent on the influx of extracellular Ca2+, which triggers a sequence of steps culminating in the restoration of PM integrity (Andrews and Corrotte, 2018). Among the better defined roles of Ca2+ influx through PM wounds are the recruitment of annexins (Swaggart et al, 2014), activation of the muscle-specific Ca2+-binding protein dysferlin (Bansal et al, 2003) and exocytosis of lysosomes at sites of PM injury (Cheng et al, 2014; Forestier et al, 2011; Ibata et al, 2019; Luisoni et al, 2015; Reddy et al, 2001). Ca2+-dependent exocytosis was initially proposed to be sufficient to promote cell resealing, by patching the wound (Terasaki et al, 1997) or reducing PM tension (Togo et al, 2000).
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